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Screening method

Inactive Publication Date: 2005-05-12
IVONEX
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0033] Moreover, the present invention provides a rest system and kit that enable compounds to be investigated for their capacity to regulate the dysregulation of the expression of members of the CEACAM family, for example to restore expression to the level of an unaltered cell. Furthermore, the test system allows the investigation of substances that influence the signal cascade via a member of the CEACAM family so that the rate of apoptosis is increased, That is to say, compounds can also be identified that, for example, through cross-linking of CEACAM molecules, contribute to a signal transmission that results in an increase in the rate of apoptosis.
[0035] In addition, the present invention relates to a diagnostic method, comprising the determination of CEACAM expression in test samples. This method permits the identification of precursor lesions at an early stage of tumorgenesis.

Problems solved by technology

For example, isolated k-ras mutations, i.e. without a preceding “gatekeeper defect”, result in the apoptosis (programmed cell death) of the affected cells.
Furthermore, no link has been described between hyperplastic lesions and dysplasia.

Method used

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Examples

Experimental program
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Effect test

example 1

CEACAM-1 Expression in Adenomas of the Colon

[0109] Freshly-derived samples of adenomas from patients were investigated for the expression of different CEACAM genes (Nollau P, et al., (1997), Cancer Res. 57, 2354-2357). For this purpose, total RNA was isolated from the samples through a conventional method and analyzed using Northern Blot (Neumaier M. et al., PNAS, (1993), 90 (22), 10744-10748).

[0110] The Northern Blots were evaluated using quantitative densitometry and image analysis and the alterations in the expression of CEACAMs compared to the corresponding normal tissue of the same patient (matched-pair analysis). The results for the adenomas are presented in Table 1 below.

TABLE 1Sample No.CEACAM1 expression 8BIØ 9BII((+))13BIØ13BII((+))14BIØ14BII((+))15BI((+))15BII(+)18BI((+))19BII((+))23BIØ23BIIØ24BI((+))25BIØ25BIIØ30B(+)31B(+)32B((+))

Ø = Specific CEACAM1 RNA no longer detectable

(+) = 60% loss in expression

((+)) = >80% loss in expression

example 2

Immunohistochemical Demonstration of the Change in CEACAM1 Expression in Different Precursor Lesions

[0111] Normal colon mucosa tissue was freshly obtained from surgical interventions. The material was fixed in 4% formaldehyde / PBS at 4° C. for 1 to 4 hours using a standard protocol. The samples were stained with 0.2% methylene blue (Sigma, Taufkirchen, Germany) for 3 minutes and investigated for the presence of ACF using a magnifying glass. Samples with more than 7 foci were used for further investigations. Samples of hyperplasic polyps, adenomas and carcinomas were obtained from the Pathology Institute of the University Hospital in Hamburg for comparison purposes.

[0112] 5 μm sections were derived from the tissue samples embedded in paraffin by a standard technique. The paraffin was removed from the sections on microscope slides and the sections washed with PBS. The sections were then pre-treated in a microwave for 10 minutes at 650 W in 10 mM citrate buffer (pH 6.0) or for 3 minut...

example 3

Significance of the Expression of CEA (CEACAM-5) for Apoptosis Sensitivity in HT29 Cells

[0123] Ribozyme-regulated mRNA expression is used to investigate the influence of CEA protein expression on the susceptibility of the cells to apoptosis. HT29 colon cancer cells (obtained from ATCC, Rockville, USA) were transfected with CEA-targeted hammerhead ribozyme expression vector.

[0124] The vector was derived by a method analogous to that of Schulte et al., PNAS 1996, 93, pages 14759ff and Juhl, H., et al, 1997, JBC 272, pages 29482ff.

[0125] The following oligonucleotides were used as ribozyme sense and antisense nucleotides.

5′-agcttTGCTCTTCTGATGAGTCCGTTAGGACGAAACTATGGAgggcc-3′ (sense)(SEQ ID NO. 1)and5′-cTCCATAGTTTCGTCCTAACGGACTCATCAGAAGAGCAa-3′ (antisense)(SEQ ID NO. 2)

[0126] These were hybridized and ligated in the pTET vector, as described by Schulte et al,, PNAS 1996, 93, pages 14759ff. The plasmid obtained, pTFT / Rz2113, contains CEA-specific flanking regions at the 5 ′ end (7 nu...

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Abstract

The invention describes a method wherein therapeutically useful compounds can be identified via the determination of the expression of glycoprotein antigens of the CEACAM family. These have properties that can prevent the development of hyperplastic alterations that have been identified as the precursors of neoplastic transformation and can lead to the development of a carcinoma, or restore the normalization of the tissue. In particular, the present invention relates to a method for the identification of one or more compounds that are suitable for the prevention of tumorigenesis or for the treatment of precursor stages of tumors. The selected compounds are in a position, through regulation of gene expression, to increase the apoptosis sensitivity (or lowers the apoptosis resistance) of cells of the colon mucosa, especially the precursor cells. In addition, the invention is directed towards a diagnostic method and towards the use of cells identified in accordance with the invention in pharmaceutical compounds for the prevention of tumorigenesis and the treatment of precursor stages.

Description

SUBJECT OF THE INVENTION [0001] The invention describes a method for the identification of one or more therapeutically useful compounds. This compound / compounds has / have characteristics that can prevent the genesis of the earliest morphological tissue alterations that can lead to the development of cancer, or to induce programmed cell death (apoptosis). As a result, the tissue situation is normalized. PRIOR ART [0002] The development of tumors, adopting colon carcinoma as an example [0003] Carcinomas develop in successive steps from normal tissue via intermediate forms (normal-to-tumor transition or multi-step carcinogenesis). Genetic alterations (mutations) have been described being related to this alteration. Multi-step carcinogenesis was formulated by the group headed by Bert Vogelstein (e.g. Fearon E. R. and Vogelstein B., Cell, 1990, 61, pages 759ff) and has been confirmed by many other groups in the literature (FIG. 1). This model states that: [0004] 1. a number of generic eve...

Claims

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Application Information

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IPC IPC(8): A61P35/00C12Q1/68C12Q1/6897G01N33/574
CPCC12Q1/6897G01N2500/00G01N33/57446A61P35/00
Inventor NEUMAIER, MICHAEL
Owner IVONEX
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